Chimeric self-sufficient P450cam-RhFRed biocatalysts with broad substrate scope

Article abstract of DOI:10.3762/bjoc.7.173

A high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of substrates was developed, leading to the identification of a number of novel biooxidation activities.

Full text of DOI:10.3762/bjoc.7.173

Chimeric self-sufficient P450cam-RhFRed
biocatalysts with broad substrate scope
Aélig Robin, Valentin Köhler, Alison Jones, Afruja Ali, Paul P. Kelly,
Elaine O'Reilly, Nicholas J. Turner and Sabine L. Flitsch*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2011, 7, 1494–1498.
School of Chemistry & Manchester Interdisciplinary Biocentre (MIB),
University of Manchester, 131 Princess Street, Manchester M1 7DN,
United Kingdom
doi:10.3762/bjoc.7.173
Received: 22 June 2011
Accepted: 19 October 2011
Published: 02 November 2011
Email:
Sabine L. Flitsch* - sabine.flitsch@manchester.ac.uk
Associate Editor: D. Trauner
* Corresponding author
© 2011 Robin et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
biocatalysis; C–H activation; high-throughput screening; P450
monooxygenase; substrate engineering
Abstract
A high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of
substrates was developed, leading to the identification of a number of novel biooxidation activities.
Introduction
P450 monooxygenases are a ubiquitous family of enzymes and others have recently reported highly active chimeric P450s
found in a wide variety of organisms in all domains of life. in which the P450 domain of a variety of bacterial enzymes is
These enzymes catalyse oxidation reactions such as hydroxyla- fused with the reductase domain of the self-sufficient P450RhF
tion, epoxidation, N- and O-dealkylation and heteroatom oxi- [12-14].
dation, often with high regio- and stereoselectivity [1], and are
therefore attractive candidates for biocatalyst development. Given that these chimeric enzymes can function in bacterial
However, the need for reconstitution of protein redox partners, hosts, we proposed that such whole-cell P450 systems would be
the necessary use of expensive NAD(P)H, and the lack of amenable to incorporation into a high-throughput screening
widely applicable high-throughput screening protocols render protocol in multiwell plates. Figure 1 outlines the design of the
the development of efficient P450 biocatalysts a major chal- screening platform: E.coli hosts containing a variety of easily
lenge. In recent years, naturally fused P450 enzymes, such as engineered chimeric constructs are incubated with the substrate
P450BM-3 [2-7] and P450RhF [8-11], have attracted great under physiological conditions, and the oxidation products
interest, although they represent only a small fraction of the are then analysed by standard analytical methods, such as
thousands of naturally occurring P450 enzymes in nature. We GC–MS.
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Beilstein J. Org. Chem. 2011, 7, 1494–1498.
Figure 1: Oxidation reactions with P450cam-RhFRed mutants.
Results and Discussion
In addition to Y96A, mutations Y96F and Y96F/V247A were
Our previous work showed that the chimera P450cam-RhFRed incorporated into the P450cam-RhFRed system for investi-
allows 100% conversion of camphor to 5-exo-hydroxycamphor gation, having shown interesting broad substrate ranges in
at 3 mM substrate concentration. The substrate specificity of previous studies with nonfused P450cam.
P450cam can be significantly broadened by specific mutations,
in particular by mutating the tyrosine residue at position 96 Initially, the microtitre-plate system was optimised and shown
(Y96) in combination with other sites such as V247 [15-22]. to give similar yields to our previously reported biotransforma-
Exchanging Y96 for an alanine residue allows the oxidation of tions. The three fused P450cam-RhFRed variants were screened
unnatural substrates, presumably because it opens up the active against a panel of substrates, which were selected on the basis
site. S. Flitsch, L.-L Wong and coworkers for example showed of the potential application of their oxidation products. For
that P450cam (Y96A) was able to hydroxylate diphenyl- instance, the terpenes α- and β-ionone and their derivatives have
methane to 4-hydroxydiphenylmethane, an activity that was not been widely used for the synthesis of carotenoids or in the
observed with the wild-type enzyme [15]. For the present study, fragrance industry [23-26], and functionalized aliphatic hetero-
incorporation of the Y96A mutation into our P450cam-RhFRed cycles are versatile building blocks for further elaboration, e.g.,
fused system made the whole cell biotransformation of diphen- by biocatalytic means [27].
ylmethane as a substrate possible (4 mL reaction in a
15 mL Falcon tube) with a GC–MS yield of 58% of 4-hydroxy- The three P450cam-RhFRed mutants were expressed in E. coli
diphenylmethane after 48 h (Scheme 1).
as previously described [12-14] and their presence confirmed by
SDS–PAGE (see Supporting Information File 1). The screening
In order to improve screening throughput, we investigated the was carried out at 4 °C in 48-well plates with a reaction volume
whole cell biotransformation of diphenylmethane with of 1 mL (180 mg wet cells/mL phosphate buffer, pH 7.2) and a
P450cam(Y96A)-RhFRed in multiwell plates (24, 48 and 96) substrate concentration of 1 mM. After 24 h and 48 h aliquots
with different reaction volumes (0.5 mL, 1 mL and 2 mL). The of the reaction mixture were extracted with ethyl acetate and
biotransformation carried out in a 48-well plate with a reaction analysed by GC–MS. In the screening process, previously
volume of 1 mL was found to give a similar conversion to the undescribed biocatalytic activities were discovered (Table 1).
4 mL reaction in a Falcon tube, and this format was therefore Thus, the monoterpenes 1, 3 and 5 were hydroxylated by all
adopted for subsequent screening of P450cam-RhFRed variants. three P450cam-RhFRed mutants, with the double mutant
Scheme 1: Whole cell biotransformation of diphenylmethane to 4-hydroxydiphenylmethane with P450cam(Y96A)-RhFRed.
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Beilstein J. Org. Chem. 2011, 7, 1494–1498.
Table 1: Conversion (% relative peak-area by GC–MS of extracted reaction mixtures), after 24 h, of substrates 1, 3, 5, and, after 48 h, of substrates 7,
11, 13 and 15 with P450cam-RhFRed mutants in 48-well plate biotransformations.
Compounds
Products
P450cam-RhFRed mutants
Substrate
Substrate no.
Y96A
>99
Y96F
Y96F/V247A
1
>99
>99
>99
1
2
4
6
3
5
94
64
98
50
3
5
>99
7a
7b
12 (8a)
48 (8b)
—
—
26 (8b)
73 (8b)a
7 (8c)
59 (10c)
31 (10c')
16 (8c)
34 (10c)
7c
85 (10c)b
7
7d
n.c.c
n.c.c
—
n.c.c
—
10
11a
<2 (u)d
9 (12b)
15 (12b')
11b
11c
11d
12 (12b)
<2 (u)e
<2 (u)d
n.c.c
11 (12c)
11
12
22 (12d)
69 (u)d
18 (12d)
82 (u)d
88 (12d)f
18 (14d)
6 (14d')
13
n.c.c
n.c.c
13
14
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Beilstein J. Org. Chem. 2011, 7, 1494–1498.
Table 1: Conversion (% relative peak-area by GC–MS of extracted reaction mixtures), after 24 h, of substrates 1, 3, 5, and, after 48 h, of substrates 7,
11, 13 and 15 with P450cam-RhFRed mutants in 48-well plate biotransformations. (continued)
15 (16d)
9 (16d')
11 (16d)
10 (16d')
15 (16d)
75 (16d')
15
15
16
aOne other product was detected (8%). bOne other product was detected (10%). cn.c. = no conversion. du = unidentified product. eTwo unidentified
products. fThree other minor products were detected (12%).
Y96F/V247A showing >99% conversion after 24 h (only one unidentified products also detected (12%). The other two
hydroxylated product was detected in the extracts).
mutants also showed high hydroxylation activity with trifluoro-
acetyl-protected 11.
Epoxidation of tetrahydropyridine 7a to compound 8a with
P450cam(Y96A)-RhFRed was shown to occur with low conver- Similar results were obtained with cyclopentylamine and cyclo-
sion (12%) with carboxybenzyl as a protecting group. In order heptylamine derivatives 13 and 15. In the case of compound 13,
to optimise the epoxidation reaction further, the N-protection only the mutant P450cam(Y96F/V247A) was able to hydroxyl-
group was used as another variable in the screening and a ate the ring, and this gave a mixture of two hydroxylated
variety of N-protecting groups were installed to generate a set products. Compound 15 was also shown to give two hydroxyl-
of tetrahydropyridine derivatives 7. An increase in conversion ated products, again with the mutant P450cam(Y96F/V247A)
(48%) was obtained with tert-butyloxycarbonyl (Boc, com- showing the highest conversions. The regioselectivity of the
pound 7b) instead of benzyloxycarbonyl (Cbz, compound 7a) hydroxylation of compounds 11, 13 and 15 has not yet been
protection. In some product mixtures from 7b additional peaks determined and is currently under investigation.
of hydroxylated compounds were observed, which could corres-
pond to structures 9, but these were not further characterised. Conclusion
Surprisingly, compound 7c, bearing a protecting group very In summary, we have described a fast screening method that
similar to Boc, showed mainly hydroxylation of an unactivated allows us to find new biocatalysts for oxidation reactions, either
carbon atom of the protecting group. Mutant P450cam(Y96F)- by screening P450 variants or by screening substrate variants
RhFRed displayed similar results to the Y96A mutant, albeit through variation of the protecting group. The combination of
with lower conversions. The double mutant P450cam(Y96F/ these reaction parameters allowed for the optimisation of a
V247A)-RhFRed turned out to be the most efficient for the specific biocatalytic reaction. The biocatalysts are very easy to
epoxidation of the tetrahydropyridine ring, with substrate 7b generate as bacterial whole-cell systems and the screening
epoxidised to 8b in 73% conversion. Racemic epoxide 8b was protocol should make this method attractive for the further
synthesised to confirm the structure of the biotransformation discovery and development of new oxidation activity.
product and to determine the enantiomeric excess. This was
found to be 18% for the biotransformation reaction with
Supporting Information
P450cam(Y96A)-RhFRed, 13% with P450cam(Y96F)-RhFRed
and 15% with P450cam(Y96F/V247A)-RhFRed.
The Supporting Information features the full experimental
procedures, characterisation of the synthesised, previously
Similar studies were performed with compound 11 in order to
unknown compounds, GC–MS traces of the
find the combination of P450 mutant and protecting group with
biotransformation reactions and the MS traces of the
the highest hydroxylation conversion. Low conversions or
corresponding starting materials.
mixtures of two products were obtained with compounds 11a,
Supporting Information File 1
11b and 11c. Interestingly, when the trifluoroacetyl protecting
group was used (11d) no starting material was detected in the
EtOAc extract of the reaction mixture after 48 h. The mutant
P450cam(Y96A)-RhFRed was shown to convert 11d to one
major hydroxylated product (12d, 88%), with three minor
Experimental part and GC–MS traces.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-7-173-S1.pdf]
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Beilstein J. Org. Chem. 2011, 7, 1494–1498.
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Acknowledgements
We wish to thank the Centre of Excellence for Biocatalysis,
Biotransformations and Biocatalytic Manufacture (CoEBio3)
and the Royal Society (Wolfson Merit Award to SLF) for finan-
cial support.
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